KR20230005257A - Anti-tumor combination containing anti-CEACAM5 antibody conjugate and FOLFIRI - Google Patents

Abstract

The present invention relates to antibody-conjugates comprising anti-CEACAM5-antibodies used to treat cancer in combination with folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI). The present invention also relates to a pharmaceutical composition and a kit of parts comprising an anti-CEACAM5-antibody in combination with folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI) used to treat cancer.

Description

Anti-tumor combination containing anti-CEACAM5 antibody conjugate and FOLFIRI

The present invention relates to antibody-conjugates comprising anti-CEACAM5-antibodies used to treat cancer in combination with folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI). The present invention also relates to a pharmaceutical composition and a kit of parts comprising an anti-CEACAM5-antibody in combination with folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI) used to treat cancer.

Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. CEA is a protein normally expressed by the fetal juvenile tract during the first six months of pregnancy and found in pancreatic, liver and colon cancers, first identified in 1965 (Gold and Freedman, J Exp Med, 121, 439, 1965 ]). The CEA family belongs to the immunoglobulin superfamily. The CEA family of 18 genes is subdivided into two sub-groups of proteins: the Carcinoembryonic Antigen-Associated Cell Adhesion Molecule (CEACAM) sub-group and the pregnancy-specific glycoprotein sub-group (Kammerer & Zimmermann, BMC Biology 2010, 8:12]).

In humans, the CEACAM sub-group consists of 7 members (CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8). A number of studies have shown that CEACAM5, identical to the originally identified CEA, is elevated on the surface of colorectal tumor cells, gastric tumor cells, lung tumor cells, breast tumor cells, prostate tumor cells, ovarian tumor cells, cervical tumor cells and bladder tumor cells. expressed in a small number of normal epithelial tissues, such as columnar epithelial cells and goblet cells of the colon, throat mucus cells of the stomach, and squamous epithelial cells of the esophagus and cervix (Hammarstrom et al., 2002, in "Tumor markers, Physiology, Pathobiology, Technology and Clinical Applications" Eds. Diamandis E.P. et al., AACC Press, Washington pp 375). Thus, CEACAM5 may constitute a suitable therapeutic target for tumor-specific targeting approaches, such as immunoconjugates.

The extracellular domains of CEACAM family members consist of repeated immunoglobulin-like (Ig-like) domains, classified according to sequence homology into three types: A, B and N. CEACAM5 contains seven such domains: N, A1, B1, A2, B2, A3 and B3. The CEACAM5 A1, A2 and A3 domains on the one hand and the B1, B2 and B3 domains on the other hand show high sequence homology, with the A domains of human CEACAM5 showing pairwise sequence similarity of 84-87% and the B domains 69 to 80% pairwise sequence similarity. Furthermore, other human CEACAM members that exhibit A and/or B domains in their structure, namely CEACAM1, CEACAM6, CEACAM7 and CEACAM8, show homology to human CEACAM5. In particular, the A and B domains of the human CEACAM6 protein show sequence homology with any of the A1 and A3 domains and the B1 to B3 domains of human CEACAM5, respectively, which is among the A and B domains of human CEACAM5. much higher than observed.

A number of anti-CEA antibodies have been generated for diagnostic or therapeutic purposes targeting CEA. As in the example by Sharkey et al. (1990, Cancer Research 50, 2823), specificity for relevant antigens is always mentioned as a concern in this field. Because of the homology mentioned above, some of the previously described antibodies may show binding to repetitive epitopes of CEACAM5 present in different immunoglobulin domains and/or other CEACAMs such as CEACAM1, CEACAM6, CEACAM7, or CEACAM8. It exhibits cross-reactivity to its members, but may lack specificity for CEACAM5. The specificity of anti-CEACAM5 antibodies is desirable in terms of CEA-targeted therapies that bind to tumor cells that express human CEACAM5 but not to some normal tissues that express other CEACAM members. CEACAM1, CEACAM6 and CEACAM8 have been described to be expressed by neutrophils of humans and non-human primates (Ebrahimmnejad et al, 2000, Exp Cell Res , 260, 365; Zhao et al, 2004, J Immunol Methods). 293, 207];

An international patent application published as WO 2014/079886 discloses antibodies that bind to the A3 to B3 domains of human and Macaca fascicularis CEACAM5 proteins, which include human CEACAM1, human CEACAM6, human CEACAM7, and human CEACAM8. , does not significantly cross-react with Macaca fascicularis CEACAM1, Macaca fascicularis CEACAM6, and Macaca fascicularis CEACAM8. This antibody was conjugated to maytansinoids to provide immunoconjugates with significant cytotoxic activity against MKN45 human gastric cancer cells with an IC ₅₀ value of 1 nM or less.

Antibody-immunoconjugates consist of an antibody attached to a cytostatic drug. In one embodiment, the antibody is attached to the cytostatic drug via a chemical linker. These immunoconjugates have great potential in cancer chemotherapy, enabling selective delivery of potent cytostatic agents targeting cancer cells, resulting in improved efficacy, reduced systemic toxicity and improved pharmacokinetics compared to conventional chemotherapy; results in pharmacodynamics and biodistribution. To date, hundreds of different immunoconjugates have been developed for a variety of cancers, several of which have been approved for use in humans.

Today, most chemotherapeutic regimens target the combined administration of cytotoxic drugs, each drug having a different mechanism of action and advantageously having a synergistic effect, causing cancer cell death. Such chemotherapy regimens are typically defined by the cytotoxic drug used, its dosage, frequency and duration of administration. Over the decades, new chemotherapeutic regimens have been developed and existing chemotherapeutic regimens are being improved for cancer treatment.

However, according to the World Health Organization, cancer was the second leading cause of death worldwide for approximately 9.6 million people in 2018. Accordingly, there is a continuing need to provide improved drug combinations and therapies for the treatment of cancer.

The present invention relates to immunoconjugates comprising an anti- CEACAM5 -antibody used in combination with folinic acid, 5- fluoro -uracil and irinotecan (FOLFIRI) to treat cancer.

The present invention also relates to an immunoconjugate comprising an anti-CEACAM5-antibody and a pharmaceutical composition comprising folinic acid, 5-fluoro-uracil and irinotecan, and the use of said pharmaceutical composition in the treatment of cancer.

In addition, the present invention provides (i) a pharmaceutical composition comprising an immunoconjugate comprising an anti-CEACAM5-antibody and (ii) one or more agents comprising folinic acid, 5-fluoro-uracil and irinotecan in separate or combined formulations. kits comprising the scientific composition(s).

The invention also relates to the use of the kit in the treatment of cancer.

Although not all possible combinations of cytostatic agents clearly show a further improved therapeutic effect, the present inventors specifically found that an immunoconjugate comprising an anti-CEACAM5-antibody in combination with FOLFIRI can be treated with either the anti-CEACAM5-antibody or FOLFIRI alone. It was found that it exhibits advantageous activity in the treatment of cancer compared to administration.

Figure 1: Activity of the immunoconjugate huMAb2-3-SPDB-DM4 and FOLFIRI therapy as single agents or in combination against the subcutaneous colon patient-derived xenograft (PDX) CR-IGR-0007P PDX in SCID mice. Changes in tumor volume by treatment group. The curve represents the median + or - MAD (median absolute deviation) for each day for each group.
Figure 2: Activity as single agent or combination of immunoconjugate huMAb2-3-SPDB-DM4 and FOLFIRI therapy against subcutaneous colon patient-derived xenograft CR-IGR-0011C PDX in SCID mice. Changes in tumor volume by treatment group. The curve represents the median + or - MAD for each day for each group.

Justice

An “ antibody ” may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (l) and kappa (k). There are five major heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains a distinct sequence domain. A light chain comprises two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains, one variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine binding recognition and specificity to antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain binding, secretion, transplacental mobility, complement binding and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable region. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. Antibody binding sites consist primarily of residues from hypervariable regions or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) affect the overall domain structure and thus the binding site. Thus, a complementarity determining region or CDR refers to an amino acid sequence that together defines the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. Each of the light chain and heavy chain of immunoglobulin has three CDRs denoted as CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H, respectively. Thus, a typical antibody antigen binding site comprises six CDRs, including sets of CDRs from each of the heavy and light chain V regions.

“ Framework regions ” (FR) refer to the amino acid sequences intervening between CDRs, ie, the portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. Each of the light and heavy chains of an immunoglobulin has four FRs denoted as FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively. Human framework regions are framework regions that are substantially identical (at least about 85%, particularly 90%, 95%, 97%, 99% or 100%) to the framework regions of a naturally occurring human antibody.

In the context of the present invention, CDR/FR definitions in an immunoglobulin light or heavy chain should be determined based on the IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77 ]; www.imgt.org).

As used herein, the term " antibody " refers to conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, particularly variable heavy chains of single domain antibodies and chimeric, humanized, bispecific or multispecific antibodies. it means.

Also, as used herein, an antibody or immunoglobulin includes “ single domain antibodies ”, which have been described more recently and are antibodies in which the complementarity determining region is part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four chain antibodies, and genetically engineered single domain antibodies. Single domain antibodies can be from any species, including but not limited to mouse, human, camel, llama, goat, rabbit, cow. A single domain antibody can be a naturally occurring single domain antibody, also known as a heavy chain antibody without a light chain. In particular, camelidae species such as camels, dromedaries, llamas, alpacas and guanacos naturally produce heavy chain antibodies devoid of light chains. Camelid heavy chain antibodies also lack the CH1 domain.

The variable heavy chains of these single domain antibodies without a light chain are known in the art as " VHH " or " nanobodies ". Like a conventional VH domain, VHH contains four FRs and three CDRs. Nanobodies have advantages over conventional antibodies. Nanobodies are about 10 times smaller than IgG molecules, and as a result, properly folded, functional nanobodies can be generated by in vitro expression, achieving high yields. Furthermore, nanobodies are very stable and resistant to the action of proteolytic enzymes. Characterization and generation of nanobodies were reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov;77(1):13-22).

As used herein, the term " monoclonal antibody " or "mAb" refers to an antibody molecule of a single amino acid sequence, which is directed against a specific antigen and is not to be construed as requiring production of the antibody by any particular method. don't Monoclonal antibodies can be produced by a single clone or hybridoma of B cells, but can also be recombinant. That is, it may be produced by protein engineering.

The term " humanized antibody " refers to an antibody that is wholly or partially of non-human origin and has been modified to replace certain amino acids, particularly in the framework regions of the VH and VL domains, to avoid or minimize an immune response in humans. The constant domains of humanized antibodies are in most cases human CH and CL domains.

A " fragment " of a (conventional) antibody comprises a portion of an intact antibody, particularly the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. included Fragments of conventional antibodies may also be heavy chain antibodies or single domain antibodies such as VHH.

The term " Fab " refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, wherein about half of the N-terminal side of the heavy chain and the entire light chain are linked together via disulfide bonds. It is generally obtained among fragments by treating IgG with a proteolytic enzyme, such as papain.

The term "F(ab')2" refers to an antibody fragment with a molecular weight of about 100,000 and an antigen-binding activity slightly greater than two identical Fab fragments linked via a disulfide bond in the hinge region. It is usually obtained among fragments by treating IgG with a proteolytic enzyme such as pepsin.

The term "Fab'" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, obtained by cleavage of the disulfide bonds in the hinge region of F(ab')2.

Single-chain Fv (" scFv ") polypeptides are covalently linked VH::VL heterodimers, which are commonly expressed from gene fusions comprising VH and VL encoding genes joined by a peptide-encoding linker. The human scFv fragments of the present invention contain CDRs maintained in proper structure, particularly using genetic recombination techniques. Bivalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs, or can be produced by linking monovalent scFvs by a peptide linker such as a divalent sc(Fv)2. "dsFv" is a VH::VL heterodimer stabilized by disulfide bonds. “(dsFv)2” means two dsFvs joined by a peptide linker.

The term " bispecific antibody " or "BsAb" refers to an antibody that combines the antigen binding sites of two antibodies in a single molecule. Thus, BsAbs can bind two different antigens simultaneously. As described for example in EP 2 050 764 A1, genetic engineering is increasingly being used to design, modify and create antibodies or antibody derivatives with a desired set of binding properties and effector functions.

The term " multispecific antibody " refers to an antibody that combines the antigen binding sites of two or more antibodies in a single molecule.

The term “ diabodies ” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) of the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains of the same chain, these domains are forced to pair with the complementary domains of the other chain, creating two antigen binding sites.

An amino acid sequence " at least 85% identical to a reference sequence " is at least 85%, in particular, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the full length of the reference amino acid sequence. , 97%, 98% or 99% sequence identity.

The percentage of " sequence identity " between amino acid sequences can be determined by comparing two sequences, optimally two sequences aligned over a comparison window, wherein a portion of the polynucleotide or polypeptide sequence within the comparison window is the best fit of the two sequences. An alignment may contain additions or deletions (i.e., gaps) when compared to a reference sequence (which does not contain additions or deletions). This percentage is determined by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and dividing the result by 100. It is calculated by multiplying to get the percentage of sequence identity. Optimal alignment of sequences for comparison is performed using global pairwise alignment, for example the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)). The percentage of sequence identity can be readily determined using the program Needle and the following parameters gap-open=10, gap-extend=0.5, for example with the BLOSUM62 matrix.

A " conservative amino acid substitution " is one in which an amino acid residue is replaced with another amino acid residue having a side chain R group with similar chemical properties (eg, charge, size, and hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of a protein. Examples of groups of amino acids having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur containing side chains: cysteine and methionine. Conservative amino acid substitutions may also be defined on the basis of amino acid size.

" Purified " and " isolated " when referring to a polypeptide (ie, antibody of the invention) or nucleotide sequence, means the presence of the indicated molecule in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" means that at least 75%, 85%, 95%, or 98% by weight of biological macromolecules of the same type are present. An “isolated” nucleic acid molecule that encodes a polypeptide of interest refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest. However, these molecules may contain some additional bases or moieties that do not adversely affect the basic properties of the composition.

As used herein, the term “ subject ” refers to mammals such as rodents, cats, dogs and primates. In particular, the subject according to the present invention is a human.

Immunoconjugates comprising anti-CEACAM5-antibodies

The present invention relates to immunoconjugates comprising an anti-CEACAM5-antibody used in combination with folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI) to treat cancer.

Immunoconjugates typically include an anti-CEACAM5-antibody and at least one cytostatic agent. In particular, in an immunoconjugate, an anti-CEACAM5-antibody is covalently attached to at least one cytostatic agent via a cleavable or non-cleavable linker.

Anti-CEACAM5-antibody

According to one embodiment, the immunoconjugate comprises a humanized anti-CEACAM5-antibody.

According to one embodiment, the immunoconjugate comprises CDR-H1 consisting of SEQ ID NO: 1, CDR-H2 consisting of SEQ ID NO: 2, CDR-H3 consisting of SEQ ID NO: 3, CDR consisting of SEQ ID NO: 4 -L1, CDR-L2 consisting of the amino acid sequence NTR, and CDR-L3 consisting of SEQ ID NO: 5.

In a further embodiment, the immunoconjugate comprises an anti-CEACAM5-antibody comprising a heavy chain variable domain (VH) consisting of SEQ ID NO: 6 and a light chain variable domain (VL) consisting of SEQ ID NO: 7.

Immunoconjugates in further embodiments

- sequence EVQLQESGPGLVKPGGSLSL

SCAAS GFVFSSY DMSWVRQTPERGLEWVAY ISSGGGIT YAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYC AAHYFGSSGPFAY WGQGTLVTVSS (SEQ ID NO: 6, CDRs shown in bold) heavy chain variable domain, wherein FR1-H covers amino acid positions 1 to 25, and CDR1-H covers amino acid positions 26 to 33 (SEQ ID NO: 1), FR2-H covers amino acid positions 34 to 50, and CDR2-H covers amino acids 51 to 58 position (SEQ ID NO: 2), FR3-H covers amino acid positions 59 to 96, CDR3-H covers amino acid positions 97 to 109 (SEQ ID NO: 3), FR4-H is a heavy chain variable domain covering amino acid positions 110 to 120, and

- the variable domain of the light chain consisting of the sequence DIQMTQSPASLSASVGDRVTITCRAS ENIFSY LAWYQQKPGKSPKLLVY NTR TLAEGVPSFSGSGSGTDFSLTISSLQPEDFATYYC QHHYGTPFT FGSGTKLEIK (SEQ ID NO: 7, CDRs in bold), wherein FR1-L covers amino acid positions 1 to 26, and CDR1 -L covers amino acid positions 27 to 32 (SEQ ID NO: 4), FR2-L covers amino acid positions 33 to 49, and CDR2-L covers amino acid positions 50 to 52 FR3-L covers amino acid positions 53 to 88, CDR3-L covers amino acid positions 89 to 97 (SEQ ID NO: 5), FR4- L includes an anti-CEACAM5-antibody, comprising the variable domain of the light chain spanning amino acid positions 98 to 107.

In a further embodiment, the immunoconjugate comprises a variable domain (VH) of a heavy chain having at least 90% identity to SEQ ID NO:6, and a variable domain (VL) of a light chain having at least 90% identity to SEQ ID NO:7. Also included is an anti-CEACAM5-antibody comprising: wherein CDR1-H consists of SEQ ID NO: 2, CDR2-H consists of SEQ ID NO: 3, and CDR3-H consists of SEQ ID NO: 4. CDR1-L consists of SEQ ID NO: 6, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 7.

In a further embodiment, the immunoconjugate comprises a variable domain (VH) of a heavy chain having at least 92%, at least 95%, at least 98% identity to SEQ ID NO:6, and at least 92%, at least to SEQ ID NO:7 An anti-CEACAM5-antibody comprising a variable domain (VL) of a light chain having 95%, at least 98% identity, wherein CDR1-H consists of SEQ ID NO: 2, and CDR2-H consists of SEQ ID NO: 2: 3, CDR3-H consists of SEQ ID NO: 4, CDR1-L consists of SEQ ID NO: 6, CDR2-L consists of amino acid sequence NTR, CDR3-L consists of SEQ ID NO: consists of 7

In a further embodiment, the immunoconjugate comprises an anti-CEACAM5-antibody comprising a heavy chain (VH) consisting of SEQ ID NO: 8 and a light chain (VL) consisting of SEQ ID NO: 9.

In a further embodiment, the immunoconjugate comprises an anti-heavy chain (VH) having at least 90% sequence identity to SEQ ID NO:8 and a light chain (VL) having at least 90% sequence identity to SEQ ID NO:9. CEACAM5-antibody, wherein CDR1-H consists of SEQ ID NO: 2, CDR2-H consists of SEQ ID NO: 3, CDR3-H consists of SEQ ID NO: 4, and CDR1-L consists of SEQ ID NO: 6, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 7.

In a further embodiment, the immunoconjugate comprises a heavy chain (VH) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO:8 and at least 92%, at least 95%, at least to SEQ ID NO:9 An anti-CEACAM5-antibody comprising a light chain (VL) with 98% identity, wherein CDR1-H consists of SEQ ID NO: 2, CDR2-H consists of SEQ ID NO: 3, and CDR3- H consists of SEQ ID NO: 4, CDR1-L consists of SEQ ID NO: 6, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 7.

The anti-CEACAM5-antibody included in the immunoconjugate may also be a single domain antibody or a fragment thereof. In particular, a single domain antibody fragment may consist of a variable heavy chain (VHH) comprising the CDR1-H, CDR2-H and CDR3-H of the antibodies described above. Such antibodies may also be heavy chain antibodies, i.e., lacking a light chain, which may or may not contain a CH1 domain.

The single domain antibody or fragment thereof may also comprise the framework regions of a Camelid single domain antibody, and optionally the constant domains of a Camelid single domain antibody.

The anti-CEACAM5-antibody included in the immunoconjugate is also selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2 and diabodies. It may be an antibody fragment, particularly a humanized antibody fragment.

Furthermore, such antibodies may be bispecific or multispecific antibodies formed from antibody fragments, at least one antibody fragment being an antibody fragment according to the present invention. Multispecific antibodies are multivalent protein complexes described, for example, in EP 2 050 764 A1 or US 2005/0003403 A1.

Anti-CEACAM5-antibodies and fragments thereof included in immunoconjugates can be generated by any technique well known in the art. In particular said antibodies are produced by techniques as described below.

Anti-CEACAM5-antibodies and fragments thereof included in the immunoconjugate may be used in isolated (eg purified) form or contained in a vector such as a membrane or lipid vesicles (eg liposomes).

Anti-CEACAM5-antibodies and fragments thereof included in immunoconjugates are produced by any technique, alone or in combination, known in the art, such as, without limitation, any chemical, biological, genetic, or enzymatic technique. It can be.

By recognizing the amino acid sequence of the desired sequence, one skilled in the art can readily generate anti-CEACAM5-antibodies and fragments thereof by standard techniques for polypeptide production. For example, they are prepared using, inter alia, commercially available peptide synthesis equipment (such as that manufactured by Applied Biosystems, Foster City, Calif.) and using well-known solid-phase methods according to the manufacturer's instructions. can be synthesized. Alternatively, anti-CEACAM5-antibodies and fragments thereof can be synthesized by recombinant DNA techniques well known in the art. For example, these fragments can be obtained as DNA expression products after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector and introducing the vector into a suitable eukaryotic or prokaryotic host that will express the desired polypeptide; , it can then be isolated from it using well-known techniques.

Anti-CEACAM5-antibodies and fragments thereof can be cultured by conventional immunoglobulin purification procedures, such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. suitably separated from the medium.

Methods for generating humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, eg, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be prepared by, for example, techniques disclosed in published patent application WO2009/032661, CDR-grafting (EP 239,400; PCT Publication WO91/09967; US Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing. ) (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)) and chain shuffling (US Patent 5,565,332). can be humanized using a variety of techniques. General recombinant DNA techniques for the preparation of such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

An anti-CEACAM5-antibody Fab can be obtained by treating an antibody that specifically reacts with CEACAM5 with a proteolytic enzyme such as papain. In addition, anti-CEACAM5-antibody Fab is prepared by inserting DNA sequences encoding both chains of the anti-CEACAM5-antibody Fab into a vector for prokaryotic or eukaryotic expression, and introducing the vector into (appropriate) prokaryotic or eukaryotic cells. and expressing the Fab of the anti-CEACAM5-antibody.

Anti-CEACAM5-antibody F(ab')2 can be obtained by treating an antibody that specifically reacts with CEACAM5 with a proteolytic enzyme such as pepsin. Alternatively, F(ab')2 of an anti-CEACAM5-antibody can be generated by linking Fab' described below via a thioether bond or a disulfide bond.

Fab' of the anti-CEACAM5-antibody can be obtained by treating F(ab')2 that specifically reacts with CEACAM5 with a reducing agent such as dithiothreitol. In addition, the Fab' of the anti-CEACAM5-antibody is expressed by inserting DNA sequences encoding the Fab' chain of the antibody into a vector for prokaryotic or eukaryotic expression, and introducing the vector into (appropriate) prokaryotic or eukaryotic cells. It can be created by doing

The scFv of the anti-CEACAM5-antibody takes the sequences of the CDR or VH and VL domains described above, constructs DNA encoding the scFv fragment, inserts this DNA into a prokaryotic or eukaryotic expression vector, and then converts this expression vector into ( It can be generated by introducing the scFv into an appropriate) prokaryotic or eukaryotic cell. To generate humanized scFv fragments, one can use the well-known technique of CDR grafting, in which complementarity determining regions (CDRs) according to the present invention are selected and placed on a human scFv fragment framework of known three-dimensional structure. (See, eg, W098/45322; WO 87/02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

cytostatic

Immunoconjugates used in accordance with the present invention typically include at least one cytostatic agent. A cytostatic agent as used herein refers to an agent that kills cells, including cancer cells. Such agents advantageously reduce tumor size by stopping cancer cells from dividing and growing. The term “cytostatic agent” is used interchangeably herein with the terms “chemotherapeutic agent,” “cytotoxic agent,” or “cytostatic drug.”

In a further embodiment, the cytostatic agent is selected from the group consisting of radioactive isotopes, protein toxins, small molecule toxins, and combinations thereof.

Radioactive isotopes are radioactive isotopes suitable for cancer treatment. Generally, such radioactive isotopes emit mainly beta-radiation. In a further embodiment, the radioactive isotope is At ²¹¹ , Bi ²¹² , Er ¹⁶⁹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , In ¹¹¹ , P ³² , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Sr ⁸⁹ , a radioactive isotope of Lu. , and combinations thereof. In one embodiment, the radioactive isotope is an alpha emitting isotope, more specifically Th ²²⁷ that emits alpha radiation.

In a further embodiment, the small molecule toxin is selected from antimetabolites, DNA-alkylating agents, DNA-crosslinking agents, DNA-intercalating agents, microtubule inhibitors, topoisomerase inhibitors, and combinations thereof.

In a further embodiment, the microtubule inhibitor is selected from the group consisting of taxanes, vinca alkaloids, maytansinoids, colchicine, podophyllotoxin, gruseofulvin, and combinations thereof.

In a further embodiment, the maytansinoid is selected from maytansinol, maytansinol analogs, and combinations thereof.

Examples of suitable analogs of maytansinol include those with altered aromatic rings and those with modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patents 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Specific examples of suitable analogs of maytansinol with a modified aromatic ring include:

(1) C-19-dechloro (US Pat. No. 4,256,746) (prepared by LAH reduction of ansamitosine P2);

(2) C-20-hydroxy (or C-20-demethyl) +/- C-19-dechloro (US Pat. Nos. 4,361,650 and 4,307,016) (demethylation using Streptomyces or Actinomyces or LAH) prepared by spent dechlorination); and

(3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (US Pat. No. 4,294,757) (prepared by acylation using an acyl chloride).

Specific examples of suitable analogs of maytansinol with other positional alterations include:

(1) C-9-SH (U.S. Patent 4,424,219) (prepared by the reaction of maytansinol with H2S or P2S5);

(2) C-14-alkoxymethyl (demethoxy/CH2OR) (US Pat. No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CHOH or CHOAc) (U.S. Patent 4,450,254) (manufactured by Nocardia);

(4) C-15-hydroxy/acyloxy (US Pat. No. 4,364,866) (prepared by conversion of maytansinol by Streptomyces);

(5) C-15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudiflora);

(6) C-18-N-demethyl (US Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Patent 4,371,533) (prepared by titanium trichloride/LAH reduction of maytansinol).

In a further embodiment, the cytotoxic conjugate of the present invention is a thiol-containing maytansinoid (DM1 ) is used as a cytotoxic agent. DM1 is represented by Formula I with the following structure:

[Formula I]

In a further embodiment, the cytotoxic conjugates of the invention contain a thiol-containing methylase, formally designated N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine. Tansinoid DM4 is utilized as a cytotoxic agent. DM4 is represented by Formula II of the structure:

[Formula II]

In further embodiments of the present invention, other maytansines may be used, including thiol and disulfide-containing maytansinoids that have mono- or di-alkyl substitutions on carbon atoms that contain sulfur atoms. They are maytansy with acylated amino acid side chains with acyl groups bearing hindered sulfhydryl groups at C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl. wherein the carbon atom of the acyl group bearing a thiol functional group has one or two substituents, the substituents being CH3, C2H5, a linear or branched alkyl or alkenyl.

Thus, in a further embodiment, the maytansinoid is (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) (DM1) or N2'-deacetyl-N-2 '(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4) and combinations thereof.

In a further embodiment, in an immunoconjugate, the anti-CEACAM5-antibody is covalently attached to at least one cytostatic agent via a cleavable or non-cleavable linker.

In a further embodiment, the linker is N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl (N -maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

In a further embodiment, the linker binds to a lysine residue in the Fc region of an anti-CEACAM5 antibody. In a further embodiment, the linker forms a disulfide bond or a thioether bond with maytansine.

In particular, the anti-CEACAM5-immunoconjugate can be selected from the group consisting of:

i) an anti-CEACAM5-SPDB-DM4-immunoconjugate of Formula III

[Formula III]

;

;

ii) anti-CEACAM5-sulfo-SPDB-DM4-immunoconjugate of Formula IV

[Formula IV]

;

;

and

iii) anti-CEACAM5-SMCC-DM1-immunoconjugate of Formula V

[Formula V]

.

.

In a further embodiment, the immunoconjugate of the invention comprises an anti-CEACAM5-antibody (huMAb2-3) comprising a heavy chain (VH) of SEQ ID NO: 8 and a light chain (VL) of SEQ ID NO: 9, and huMAb2 -3 to N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4) via N-succinimidyl pyridyldithiobutyrate (SPDB) connected in common Thus, the immunoconjugate huMAb2-3-SPDB-DM4 is obtained.

As used herein, “linker” refers to a chemical moiety comprising a chain or covalent bond of atoms that covalently attaches a polypeptide to a drug moiety.

These conjugates can be prepared by in vitro methods. To link a drug or prodrug to an antibody, a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Conjugation of an antibody of the present invention with a cytotoxic agent or growth inhibitory agent is N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2, 5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyl dithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g. dimethyl adipimi date HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde), bis-azido compounds (eg bis (p-azidobenzoyl)-hexanediamine), bis-dia Zonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro- 2,4-dinitrobenzene), but not limited thereto. For example, a ricin immunotoxin can be prepared as described in Vitetta et al. (1987). Carbon-labeled 1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionuclides to antibodies (WO 94/11026).

The linker may be a “cleavable linker” to facilitate release of the cytotoxic agent or growth inhibitor within the cell. For example, acid-labile linkers, peptidase-sensitive linkers, esterase-labile linkers, photolabile linkers or disulfide-containing linkers (see, eg, US Pat. No. 5,208,020) may be used. Such linkers may in some cases be “non-cleavable linkers” (eg SMCC linkers) which may result in better tolerance.

Generally, conjugates can be obtained by a method comprising the steps of:

(i) optionally contacting a buffered aqueous solution of a cell binding agent (eg an antibody according to the invention) with a solution of a linker and a cytotoxic compound;

(ii) then, optionally, separating the conjugate formed in step (i) from the unreacted cell binding agent.

Aqueous solutions of these cell binding agents may be buffered with a buffer such as, for example, potassium phosphate, acetate, citrate or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffering agent depends on the nature of the cell binding agent. Cytotoxic compounds are solutions in organic polar solvents such as, for example, dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

The reaction temperature is generally comprised between 20 and 40°C. The reaction time may vary from 1 hour to 24 hours. The reaction of the cell binding agent with the cytotoxic agent can be monitored by size exclusion chromatography (SEC) using refractometry and/or UV detectors. If the conjugate yield is too low, the reaction time may be extended.

A person skilled in the art can use a number of different chromatographic methods to carry out the separation of step (ii): the conjugate can be prepared using, for example, SEC, adsorption chromatography (eg ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed support chromatography such as hydroxyapatite chromatography, or high performance liquid chromatography (HPLC). Purification by dialysis or diafiltration may also be used.

As used herein, the term “aggregate” refers to an association that can form between two or more cell binding agents, which may or may not be altered by conjugation. Aggregates can form under the influence of a number of parameters, such as the high concentration of the cell binding agent in the solution, the pH of the solution, the high shear force, the number of bound dimers and their hydrophobic nature, temperature (Wang & Gosh, 2008, J. Membrane Sci., 318: 311-316 and references cited therein); Note that the relative influence of some of these parameters has not been clearly established. For proteins and antibodies, the skilled person will refer to Cromwell et al., 2006, AAPS Journal, 8(3): E572-E579. The content of aggregates can be determined by techniques well known to those skilled in the art, such as SEC (see Walter et al., 1993, Anal. Biochem., 212(2): 469-480).

After step (i) or (ii), the conjugate-containing solution may be subjected to an additional step (iii) of chromatography, ultrafiltration, and/or diafiltration.

The conjugate is recovered in aqueous solution at the end of these steps.

In a further embodiment, an immunoconjugate according to the invention is characterized by a “drug-to-antibody ratio” (or “DAR”) ranging from 1 to 10, 2 to 5, or 3 to 4. This is generally the case for conjugates containing maytansinoid molecules.

These DAR values depend on the properties of the drug (i.e., growth inhibitor) and antibody used together with the experimental conditions used for conjugation (such as growth inhibitor/antibody ratio, reaction time, and properties of the solvent and, if present, co-solvent). can be different. Thus, contacts between an antibody and a growth inhibitor may involve several conjugates that differ from each other by different drug-to-antibody ratios; optionally a naked antibody; optionally resulting in a mixture comprising aggregates. Therefore, the determined DAR is an average value.

A method that can be used to determine the DAR consists of spectroscopically measuring the ratio of λD and absorbance at 280 nm of a substantially purified conjugate solution. 280 nm is a wavelength commonly used to measure protein concentrations such as antibody concentrations. The wavelength λD is chosen to allow discrimination of the drug from the antibody, i.e., as readily known to those skilled in the art, λD is the wavelength at which the drug has high absorbance, and λD is used to avoid substantial overlap in the absorbance peaks of the drug and antibody. It is far enough away from 280 nm. λD can be chosen to be 252 nm for maytansinoid molecules. The DAR calculation method can be derived from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science.

The absorbance for the conjugate at λD (AλD) and 280 nm (A280) can be measured on the monomer peak of a size exclusion chromatography (SEC) analysis (which allows the calculation of the “DAR (SEC)” parameter) or on (“DAR (UV)” )" parameters) are measured using typical spectroscopic instruments. Absorbance can be expressed as:

AλD = (cD x εDλD) + (cA x εAλD)

A280 = (cD x εD280) + (cA x εA280)

in the expression:

cD and cA are the concentrations of drug and antibody in solution, respectively;

εDλD and εD280 are the molar extinction coefficients of the drug at λD and 280 nm, respectively;

εAλD and εA280 are the molar extinction coefficients of the antibody at λD and 280 nm, respectively.

Solving these two equations with two unknowns leads to the following equation:

cD = [(εA280 x AλD) - (εAλD x A280)] / [(εDλD x εA280) - (εAλD x εD280)]

cA = [A280 - (cD x εD280)] / εA280

Then, the average DAR is calculated from the ratio of the concentration of drug to the concentration of antibody: DAR = cD / cA.

FOLFIRI

An immunoconjugate comprising an anti-CEACAM5-antibody is used in combination with FOLFIRI for the treatment of cancer.

FOLFIRI itself is a known chemotherapeutic regimen approved for use in humans that includes the combined administration of folinic acid, 5-fluoro-uracil and irinotecan, which is typically administered in 2-week cycles of up to 12 doses. FOLFIRI is used in combination with drugs, each with a different mechanism of action and advantageously having a synergistic effect, causing cancer cell death.

5-Fluoro-uracil (CAS Registry No. 51-21-8) is an anti-metabolite, which primarily inhibits thymidylate synthase, thereby blocking the synthesis of thymidine. 5-Fluoro-uracil has been used for the treatment of colon, esophageal, gastric, pancreatic, breast and cervical cancers.

Folic acid, also known as leucovorin (CAS Registry No. 58-05-9), stabilizes the complex of 5-fluoro-uracil and thymidylate synthase, increasing the cytotoxicity of 5-fluoro-uracil. In one embodiment, the folinic acid is L-folinic acid (N-[4-[[[(6S)-2-amino-5-formyl-3,4,5,6,7,8-hexahydro-4- oxo-6-pteridinyl]methyl]amino]benzoyl]-L-glutamic acid). In another embodiment, the folinic acid is the calcium salt of L-folinic acid. Folic acid may also contain mixtures of two or more stereoisomers.

Irinotecan (CAS number 97682-44-5) is a cytotoxic, semi-synthetic derivative of the alkaloid camptothecin, inhibits topoisomerase I, thereby inhibiting DNA replication and transcription, and is used in the treatment of colon cancer and small cell lung cancer. come.

combination therapy

According to the present invention, an immunoconjugate comprising an anti-CEACAM5-antibody is used to treat cancer in combination with folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI). The invention also relates to folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI) used to treat cancer in combination with an immunoconjugate comprising an anti-CEACAM5-antibody.

The present invention also relates to a subject in need thereof, comprising administering an immunoconjugate comprising an anti-CEACAM5-antibody to a subject in need thereof, and further administering folinic acid, 5-fluoro-uracil and irinotecan. It relates to a method of treating cancer in a subject having.

The present invention also relates to an immunoconjugate comprising an anti-CEACAM5-antibody, which is used to treat cancer in a subject in need thereof which either individually or concurrently accepts FOLFIRI, further comprising folinic acid, 5-fluoro-uracil and irinotecan can be administered separately or simultaneously.

In one embodiment, the cancer is a solid tumor. According to one embodiment, the cancer is selected from the group consisting of colorectal cancer, gastric cancer, pancreatic cancer and esophageal cancer. In a further embodiment, the cancer is colorectal cancer.

According to one embodiment, the patient has a malignancy, particularly a malignant solid tumor, more specifically a locally advanced or metastatic solid malignancy.

According to one embodiment, an immunoconjugate comprising an anti-CEACAM5-antibody and FOLFIRI are administered concurrently to a subject in need thereof.

In a further embodiment, the immunoconjugate comprising an anti-CEACAM5-antibody and FOLFIRI are (i) in a single pharmaceutical composition comprising the immunoconjugate and FOLFIRI, or (ii) at least one pharmaceutical composition comprises an anti-CEACAM5-antibody wherein the one or more pharmaceutical compositions are formulated in the form of at least two separate pharmaceutical compositions comprising folinic acid, 5-fluoro-uracil and irinotecan in separate or combined formulations. In the case of formulations of the immunoconjugate and FOLFIRI of at least two separate pharmaceutical compositions, the at least two separate pharmaceutical compositions are administered simultaneously to a subject in need thereof.

According to another embodiment, the immunoconjugate comprising an anti-CEACAM5-antibody and FOLFIRI are administered separately or sequentially to a subject in need thereof.

According to this embodiment, the immunoconjugate comprising the anti-CEACAM5-antibody and FOLFIRI are formulated in the form of at least two separate pharmaceutical compositions, wherein (i) at least one pharmaceutical composition comprises the immunoconjugate; (ii) the at least one pharmaceutical composition comprises folinic acid, 5-fluoro-uracil and irinotecan, either separately or in combination.

In one embodiment, the immunoconjugate is administered at a dose of 60 to 210 mg/m ² . In another embodiment, folinic acid is administered at a dose of 100 to 400 mg/m ² , or L-folinic acid is administered at a dose of 100 to 200 m/m ² . In another embodiment, 5-fluoro-uracil is administered at a dose of 1000 to 2000 mg/m ² . In another embodiment, irinotecan is administered at a dose of 100 to 300 mg/m ² .

In another embodiment, the pharmaceutical composition or combination of the invention is wherein the anti-CEACAM5-antibody is administered at a dose of 60 to 210 mg/m ² and the folinic acid is administered at a dose of 200 to 600 mg/m ² , or L -Folinic acid is administered at a dose of 100 to 200 m/m ² , 5-fluorouracil (5-FU) is administered at a dose of 2000 to 4000 mg/m ² , and irinotecan is administered at 100 and about 300 mg/m ² It is administered to be administered at a dose of In aspects of this embodiment, the dosing regimen comprises administering the dose over a period of 2 hours to 48 hours. In aspects of this embodiment, the frequency of administration varies from once weekly to once every three weeks. In one embodiment, the treatment period is at least 4 or 6 months.

In a further embodiment, the immunoconjugate comprising an anti-CEACAM5-antibody and folinic acid or L-folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI) are administered in 8 to 16 cycles. According to one embodiment, the cycle is selected from a 1 week cycle, a 2 week cycle or a 3 week cycle. According to one embodiment, one cycle includes:

administering the immunoconjugate at a dose of 60 to 210 mg/m ² /day at least once during the cycle;

administering folinic acid at a dose of 100 to 300 mg/m ² /day or L-folinic acid at a dose of 100 to 200 m/m ² at least once during a cycle;

administering 5-fluoro-uracil at a dose of 1000 to 2000 mg/m ² at least once during the cycle, and

Administering irinotecan at a dose of 100 to 300 mg/m ² at least once during the cycle.

In one embodiment, the immunoconjugate is administered at a dose of 60 to 210 mg/m ² on cycle day 1. In one embodiment, folinic acid is administered at a dose of 100 to 300 mg/m ² or L-folinic acid is administered at a dose of 100 to 200 m/m ² on days 1 and 2 of the cycle. In one embodiment, 5-fluoro-uracil is administered at a dose of 1000 to 2000 mg/m ² on days 1 and 2 of the cycle. In one embodiment, irinotecan is administered at a dose of 100 to 300 mg/m ² on cycle day 1.

The unit “mg/m ² ” represents the amount of the compound in units of mg/m ² of the subject's body surface to be administered. One skilled in the art will know how to determine the amount of compound needed for a subject to be treated based on body surface area, which will in turn be calculated based on height and weight.

The present invention also relates to a pharmaceutical composition comprising an immunoconjugate comprising an anti-CEACAM5-antibody and further comprising folinic acid, 5-fluoro-uracil and irinotecan.

In addition, the present invention provides (i) a pharmaceutical composition comprising an immunoconjugate comprising an anti-CEACAM5-antibody and (ii) one or more agents comprising folinic acid, 5-fluoro-uracil and irinotecan in separate or combined formulations. It relates to a kit comprising a scientific composition.

The present invention also relates to a pharmaceutical composition comprising an immunoconjugate comprising an anti-CEACAM5-antibody, further comprising folinic acid, 5-fluoro-uracil and irinotecan, for use in treating cancer.

In addition, the present invention is used to treat cancer, (i) a pharmaceutical composition comprising an immunoconjugate comprising an anti-CEACAM5-antibody and (ii) folinic acid, 5-fluoro-uracil in separate or combined formulations and one or more pharmaceutical compositions comprising irinotecan.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecular components and compositions that do not produce side effects, allergic or other side reactions when administered to mammals, and particularly to humans where appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents, and/or excipients include water, amino acids, saline, phosphate buffered saline, buffered phosphate, acetate, citrate, succinate; amino acids and derivatives such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts NaCl, calcium chloride; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as polysorbate 80, polysorbate 20, poloxamer 188, and the like, and combinations thereof. In many cases, it will be desirable to include sugars, polyhydric alcohols, or tonicity agents such as sodium chloride in the composition, and the formulations may also contain antioxidants such as tryptamine and stabilizers such as Tween 20.

The form of the pharmaceutical composition, route of administration, dosage and administration plan naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient and the like.

The pharmaceutical composition of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

In particular, pharmaceutical compositions contain a pharmaceutically acceptable vehicle in an injectable form. They are, inter alia, isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts) or injectable solutions upon addition of sterile water or physiological saline as the case may be. may be a dried, particularly freeze-dried composition that allows for a composition of

Such pharmaceutical compositions may be administered through a drug combination device.

The dose employed for administration may be adjusted according to various parameters, in particular according to the mode of administration used, according to the pathology involved, or alternatively according to the desired duration of treatment.

To prepare a pharmaceutical composition, an effective amount of an immunoconjugate comprising an anti-CEACAM5-antibody and folinic acid, 5-fluoro-uracil and irinotecan can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. there is.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations comprising sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, these forms must be sterile and must be injectable with an appropriate device or system so that they can be delivered without degradation. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

Solutions of the active compounds as free bases or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under normal conditions of storage and use, these formulations contain preservatives to prevent the growth of microorganisms.

Immunoconjugates comprising an anti-CEACAM5-antibody may be formulated as a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), which are formed, for example, with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, glycine, histidine, procaine, and the like. can

The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases it will be desirable to include a tonicity agent such as sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilizing active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying techniques which produce a powder of the active ingredient and any additional required ingredients from a previously sterile filtered solution.

Preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, where using DMSO as the solvent is envisioned, resulting in extremely rapid penetration to deliver high concentrations of the active agent to small tumor areas.

When formulated, the solution will be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulation is readily administered in a variety of dosage forms, such as the injectable solution types described above, but drug release capsules and the like may also be used.

For parenteral administration in aqueous solutions, for example, such solutions should be suitably buffered as necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 ml of an isotonic NaCl solution, added to 1000 ml of subcutaneous infusion, or injected at a predetermined site of infusion (see, e.g., "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will inevitably occur depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dose for the individual subject in any case.

Immunoconjugates comprising an anti-CEACAM5-antibody are formulated for parenteral administration, such as intravenous or intramuscular injection, and other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; time-release capsules; and any other form currently in use.

In certain embodiments, the use of liposomes and/or nanoparticles is contemplated for introduction of the polypeptide into a host cell. The formation and use of liposomes and/or nanoparticles is known to those skilled in the art.

Nanocapsules can generally entrap compounds in a stable and reproducible manner. In order to avoid side effects due to intercellular polymer overload, such ultrafine particles (about 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles, or biodegradable polylactide, or polylactide co glycolide nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be readily prepared. can

Liposomes are formed from phospholipids dispersed in an aqueous medium, which spontaneously form multilamellar concentric bilayer vesicles (also referred to as multilamellar vesicles (MLVs)). MLVs generally have diameters ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å, containing an aqueous solution in the core. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Brief description of the sequence

SEQ ID NOs: 1 to 5 represent the sequences of CDR1-H, CDR2-H, CDR3-H, CDR1-L and CDR3-L of the anti-CEACAM5-antibody (huMAb2-3).

SEQ ID NO: 6 shows the sequence of the variable domain (VH) of the heavy chain of the anti-CEACAM5-antibody (huMAb2-3).

SEQ ID NO: 7 shows the sequence of the variable domain (VL) of the light chain of the anti-CEACAM5-antibody (huMAb2-3).

SEQ ID NO: 8 represents the heavy chain sequence of the anti-CEACAM5-antibody (huMAb2-3).

SEQ ID NO: 9 represents the light chain sequence of the anti-CEACAM5-antibody (huMAb2-3).

Example

Example 1: Activity of the immunoconjugate huMAb2-3-SPDB-DM4 in combination with FOLFIRI against two subcutaneous colon patient-derived xenografts CR-IGR-0007P PDX and CR-IGR-0011C PDX in SCID mice.

experimental procedure

The activity of huMAb2-3-SPDB-DM4 and FOLFIRI therapy was evaluated in two subcutaneous colon patient-derived xenografts (PDX) (CR-IGR-0007P PDX and CR-IGR-0011C) injected subcutaneously (s.c.) into female SCID mice. PDX) as a single agent or as a combination. Controls were left untreated. The dose of compound used is given in units of mg/kg.

For CR-IGR-0007P PDX, treatment was started when median tumor burden reached 166.0 mm ³ on day 26 after tumor injection. huMAb2-3-SPDB-DM4 was administered intravenously (IV) at 5 mg/kg on days 26, 33 and 40 following a cycle of 3 weeks. The FOLFIRI regimen was administered according to a 3-week cycle, consisting of intravenous administration of folinic acid at 60 mg/kg and irinotecan at 22 mg/kg on days 26, 33 and 40 and days 27 and 34. and intravenous administration of 56 mg/kg of 5-FU on day 41.

For CR-IGR-0011C PDX, treatment was started when the median tumor burden reached 123.5 mm ³ on day 19 after tumor injection. huMAb2-3-SPDB-DM4 was administered intravenously at 5 mg/kg on days 19, 26 and 33 following a cycle of 3 weeks. The FOLFIRI regimen was administered according to a 3-week cycle, consisting of intravenous administration of folinic acid at 60 mg/kg and irinotecan at 22 mg/kg on days 19, 26, and 33 and days 20, 27. and intravenous administration of 5-FU at 56 mg/kg on day 34.

For evaluation of anti-tumor activity, animals were weighed daily and tumors were measured twice weekly with calipers. A dose that resulted in a 20% weight loss or 10% or more drug deaths at the lowest point (group mean) was considered an overly toxic dose. Animal body weights included tumor weights. Tumor volume was calculated using the formula: mass (mm ³ ) = [length (mm) x width (mm) x width (mm)]/2. The primary efficacy endpoint was ΔT/ΔC, percent median regression, partial and complete regression (PR and CR).

The change in tumor volume in each treatment group (T) and control group (C) is calculated by subtracting the tumor volume on the first treatment day (staging day) from the tumor volume on the specified observation day for each tumor. A median ΔT is calculated for the treatment group and a median ΔC is calculated for the control group. The ratio ΔT/ΔC is then calculated and expressed as a percentage: ΔT/ΔC = (Delta T/Delta C) x 100.

A dose is considered therapeutically active when ΔT/ΔC is less than 40% and highly active when ΔT/ΔC is less than 10%. If ΔT/ΔC is less than zero, these doses are considered highly active and regression rates are recorded by date (see Plowman J, Dykes DJ, Hollingshead M, Simpson-Herren L and Alley MC. Human tumor xenograft models). in NCI drug development. In: Feibig HH BA, editor. Basel: Karger.; 1999 p 101-125]):

Percent tumor regression is defined as the percent reduction in tumor volume in the treatment group on the specified observation day compared to the tumor volume on the first day of primary treatment.

The percent regression for each animal at a specific time point is calculated. Then, the median % regression is calculated for that group:

Regression % (at t) =

Partial regression (PR): Partial regression is defined when the tumor volume is reduced to 50% of the tumor volume at the start of treatment.

Complete Regression (CR): Complete regression is achieved when tumor volume = 0 mm ³ (considered CR when tumor volume could not be recorded).

result

Results for CR-IGR-0007P PDX are presented in Figure 1 and Table 1 (below).

One mouse in the control group was found dead on D54; CR-IGR-0007P is an invasive tumor and may be cachexic. huMAb2-3-SPDB-DM4 was administered at less than the maximum tolerated dose (MTD) and was well tolerated and did not induce toxicity. FOLFIRI therapy was administered at its respective MTD determined in tumor-free mice. In these CR-IGR-0007P tumor-bearing mice, the cytotoxic treatment alone or in combination was compliant, with a weight loss of 8.1 to 10.8% except for one mouse in the group treated with the combination, as described above. Mice continued to lose weight until greater than 20% weight loss was reached and died on D48.

As a single agent, huMAb2-3-SPDB-DM4 was inactive, with a ΔT/ΔC of 76% on D49. FOLFIRI therapy as a single agent was highly active, with ΔT/ΔC lower than 0% (p <0.0001) and tumor regression of 18% (Table 1).

The combination of huMAb2-3-SPDB-DM4 and FOLFIRI therapy was highly active, with a ΔT/ΔC lower than 0% (p <0.0001), tumor regression of 47%, and 4 PRs (partial regression). The combined effect of huMAb2-3-SPDB-DM4 and FOLFIRI was significantly different from the effect of huMAb2-3-SPDB-DM4 alone on days 33 to 62, and was significantly different from the effect of FOLFIRI alone on days 33 to 62 were distinctly different.

In conclusion, in CR-IGR-0007P PDX, huMAb2-3-SPDB-DM4 was inactive as a single agent after intravenous administration for 3 weeks at 5 mg/kg, whereas FOLFIRI therapy was highly active and well amenable to treatment. The combination of huMAb2-3-SPDB-DM4 and FOLFIRI therapy was more active than single agents.

[Table 1]

Results for CR-IGR-0011C PDX are presented in Figure 2 and Table 2 (below).

Control mice showed negative body weight change (nadir of -6.7% on day 32); CR-IGR-0011C is an invasive tumor and may be cachexic. huMAb2-3-SPDB-DM4 was administered at less than the maximum tolerated dose (MTD) and was well tolerated and did not induce toxicity.

FOLFIRI therapy was administered with its MTD determined in tumor-free mice. In these CR-IGR-0011C tumor-bearing mice that induced weight loss, cytotoxic treatment alone or in combination induced additional weight loss, and a high-calorie dietary supplement for laboratory rodents was added to each group on D24. did FOLFIRI therapy alone or in combination resulted in weight loss of 5.6 to 9.8%.

As a single agent, huMAb2-3-SPDB-DM4 is highly active, with a ΔT/ΔC lower than 0% (p <0.0001) at D35, tumor regression of 29% and 2 PRs. FOLFIRI therapy as a single agent was highly active, with a ΔT/ΔC of 2% (p <0.0001).

The combination of huMAb2-3-SPDB-DM4 with FOLFIRI therapy was highly active, ΔT/ΔC lower than 0% (p <0.0001), tumor regression was 88%, 6 PRs and 2 CRs (complete regression). The combined effect of huMAb2-3-SPDB-DM4 and FOLFIRI was significantly different from the effect of huMAb2-3-SPDB-DM4 alone on days 22 to 35, and the effect of FOLFIRI alone on days 30 to 35 was significant. were distinctly different.

In conclusion, in the CR-IGR-0001C PDX, huMAb2-3-SPDB-DM4 was highly active as a single agent after intravenous administration at 5 mg/kg for 3 weeks. FOLFIRI was also highly active as a single agent. The combination of HUMAB2-3-SPDB-DM4 and FOLFIRI was significantly more active than the corresponding single agent.

[Table 2]

SEQUENCE LISTING <110> Sanofi <120> ANTITUMOR COMBINATIONS CONTAINING ANTI-CEACAM5 ANTIBODY CONJUGATES AND FOLFIRI <130> 89597-S266-SANOFI-HG <150> EP20315219.4 <151> 2020-04-24 <160> 9 <170> BiSSAP 1.3.6 <210> 1 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-H1 of anti-CEACAM5-antibody <400> 1 Gly Phe Val Phe Ser Ser Tyr Asp 1 5 <210> 2 <211> 8 <212> PRT <213> artificial sequence <220> <223> CDR-H2 of anti-CEACAM5-antibody <400> 2 Ile Ser Ser Gly Gly Gly Ile Thr 1 5 <210> 3 <211> 13 <212> PRT <213> artificial sequence <220> <223> CDR-H3 of anti-CEACAM5-antibody <400> 3 Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr 1 5 10 <210> 4 <211> 6 <212> PRT <213> artificial sequence <220> <223> CDR-L1 of anti-CEACAM5-antibody <400> 4 Glu Asn Ile Phe Ser Tyr 1 5 <210> 5 <211> 9 <212> PRT <213> artificial sequence <220> <223> CDR-L3 of anti-CEACAM5-antibody <400> 5 Gln His His Tyr Gly Thr Pro Phe Thr 1 5 <210> 6 <211> 120 <212> PRT <213> artificial sequence <220> <223> Variable domain of heavy chain of anti-CEACAM5-antibody <400> 6 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Arg Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ile Thr Tyr Ala Pro Ser Thr Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 7 <211> 107 <212> PRT <213> artificial sequence <220> <223> Variable domain of light chain of anti-CEACAM5-antibody <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Asn Thr Arg Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 8 <211> 449 <212> PRT <213> artificial sequence <220> <223> Heavy chain of anti-CEACAM5-antibody <400> 8 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Arg Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ile Thr Tyr Ala Pro Ser Thr Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Tyr Phe Gly Ser Ser Gly Pro Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <210> 9 <211> 214 <212> PRT <213> artificial sequence <220> <223> Light chain of anti-CEACAM5-antibody <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Asn Thr Arg Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims (22)

An immunoconjugate comprising an anti-CEACAM5-antibody for use in combination with folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI) to treat cancer. According to claim 1,
The anti-CEACAM5-antibody comprises CDR-H1 consisting of SEQ ID NO: 1, CDR-H2 consisting of SEQ ID NO: 2, CDR-H3 consisting of SEQ ID NO: 3, CDR-L1 consisting of SEQ ID NO: 4 , CDR-L2 consisting of the amino acid sequence NTR, and CDR-L3 consisting of SEQ ID NO: 5. According to claim 1 or 2,
wherein the anti-CEACAM5-antibody comprises a heavy chain variable domain (VH) consisting of SEQ ID NO: 6 and a light chain variable domain (VL) consisting of SEQ ID NO: 7. According to any one of claims 1 to 3,
wherein the anti-CEACAM5-antibody comprises a heavy chain (VH) consisting of SEQ ID NO: 8 and a light chain (VL) consisting of SEQ ID NO: 9. According to any one of claims 1 to 4,
An immunoconjugate comprising at least one cytostatic agent. According to claim 5,
wherein the cell proliferation inhibitor is selected from the group consisting of radioactive isotopes, protein toxins, small molecule toxins, and combinations thereof. According to claim 6,
wherein the small molecule toxin is selected from antimetabolites, DNA-alkylating agents, DNA-crosslinking agents, DNA-intercalating agents, microtubule inhibitors, topoisomerase inhibitors, and combinations thereof. According to claim 7,
Wherein the microtubule inhibitor is selected from the group consisting of taxanes, vinca alkaloids, maytansinoids, colchicine, podophyllotoxin, gruseofulvin, and combinations thereof. According to claim 8,
The maytansinoid is N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or N2'-deacetyl-N-2'(4-methyl-4- An immunoconjugate selected from the group consisting of mercapto-1-oxopentyl)-maytansine (DM4) and combinations thereof. According to any one of claims 1 to 9,
wherein the anti-CEACAM5-antibody is covalently attached to the at least one cytotoxic agent through a cleavable or non-cleavable linker. According to claim 10,
The linker is N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl (N-maleimidomethyl ) cyclohexane-1-carboxylate (SMCC). According to any one of claims 1 to 11,
It includes a CEACAM5-antibody (huMAb2-3) comprising a heavy chain (VH) consisting of SEQ ID NO: 8 and a light chain (VL) consisting of SEQ ID NO: 9, which is N-succinimidyl pyridyldithiobutyrate ( SPDB) to N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). According to any one of claims 1 to 12,
An immunoconjugate characterized by a drug to antibody ratio (DAR) ranging from 1 to 10. According to any one of claims 1 to 13,
wherein the cancer is selected from the group consisting of colorectal cancer, gastric cancer, pancreatic cancer and esophageal cancer. According to any one of claims 1 to 14,
wherein the immunoconjugate and FOLFIRI are administered simultaneously to a subject in need thereof. According to claim 15,
The immunoconjugate and FOLFIRI may be (i) in a single pharmaceutical composition comprising the immunoconjugate and FOLFIRI, or (ii) at least one pharmaceutical composition comprising the immunoconjugate and one or more pharmaceutical compositions used separately or in combination. An immunoconjugate formulated in the form of at least two separate pharmaceutical compositions comprising folinic acid, 5-fluoro-uracil and irinotecan in dosage form. According to any one of claims 1 to 14,
wherein the immunoconjugate and FOLFIRI are administered separately or sequentially to a subject in need thereof. According to claim 17,
The immunoconjugate and FOLFIRI are formulated in the form of at least two separate pharmaceutical compositions, wherein (i) at least one pharmaceutical composition comprises the immunoconjugate, and (ii) one or more pharmaceutical compositions are used separately or in combination. An immunoconjugate comprising folinic acid, 5-fluoro-uracil and irinotecan in a formulation. According to any one of claims 1 to 18,
The immunoconjugate comprising an anti-CEACAM5-antibody, and folinic acid, 5-fluoro-uracil and irinotecan (FOLFIRI) are administered in 8 to 16 cycles, wherein 1 cycle is
administering said immunoconjugate at a dose of 60 to 210 mg/m ² at least once during a cycle;
administering folinic acid at a dose of 100 to 300 mg/m ² or L-folinic acid at a dose of 100 to 200 m/m ² at least once during the cycle;
administering 5-fluoro-uracil at a dose of 1000 to 2000 mg/m ² at least once during the cycle, and
and administering irinotecan at a dose of 100 to 300 mg/m ² at least once during the cycle. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 to 14 and folinic acid, 5-fluoro-uracil and irinotecan. (i) a pharmaceutical composition of the immunoconjugate of any one of claims 1 to 14 and (ii) one or more pharmaceutical compositions comprising folinic acid, 5-fluoro-uracil and irinotecan in separate or combined formulations. Do, Kit. A pharmaceutical composition according to claim 20 or a kit according to claim 21 , for use in treating cancer.

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